EA023379B1 - STRAIN ESCHERICHIA COLI BL21(DE3)-pIE-GAMMA AND METHOD FOR OBTAINING PREPARATION OF NATIVE RECOMBINANT HUMAN GAMMA INTERFERON - Google Patents

Abstract

The invention relates to medical biotechnology. Disclosed is a group of the inventions: strain Escherichia coli BL21(DE3)-pIE-gamma, being producent of human gamma interferon and a method for obtaining highly purified preparation of unprocessed methionine-free native recombinant human gamma interferon.

Description

The invention relates to medical biotechnology.

Interferon gamma is a multifunctional protein produced by the cells of the immune system (T-cells and ΝΚ-cells), has immunostimulating (activates T-cells), antitumor (antiproliferative) - due to the increase in the number of MHC I molecules and the induction of antiangiogenic effect through the secretion of interferon-dependent -gamma ΙΡ-10 and MJ - and antiviral activity - activates macrophages by increasing the number of MHC II molecules.

Natural human immune interferon (interferon gamma) is a glycoprotein with a molecular weight of 16.9 kDa, is a soluble cytokine, with p1 9.7, is synthesized as a precursor of 166 amino acid residues with a signal peptide (20 amino acid residues), contains two glycosylation sites (48 and 120 amino acid residue). Glycosylation of interferon gamma plays an important role in its secretion from the cell, when using the recombinant protein interferon gamma for therapeutic purposes during injection, it immediately enters the bloodstream, bypassing the secretion stage. Moreover, it is known that the activity of the glycosylated and non-glycosylated forms of interferon gamma is comparable (8atepea T. s1 a1., 1994). This makes it possible to use for pharmaceutical purposes the non-glycosylated recombinant protein interferon gamma obtained in bacterial E. col cells.

The creation of microorganisms producing human interferon gamma made it possible to obtain a biologically active recombinant protein in an amount necessary for conducting extensive structural and functional studies, as well as for use in medical practice. Medicines based on recombinant gamma interferon have been developed for the treatment of immune, oncological and severe viral diseases (Asyttype for the treatment of chronic granulomatosis, 1kt for the treatment of psoriasis, leprosy, rheumatoid arthritis, eczema and other diseases, Ingaron - for the treatment of chronic viral hepatitis C, chronic hepatitis B virus, HIV / AIDS infection, pulmonary tuberculosis in complex therapy, genital herpes virus infection and herpes zoster (Negre8 / 051eg) in monotherapy, urogenital chl amidiosis in complex therapy, oncological diseases in complex therapy as an immunomodulator, including in combination with chemotherapy, for the prevention of infectious complications in patients with chronic granulomatous disease, etc.). However, one should strive to reduce the number and severity of side effects from therapy with human interferon gamma in the above drugs. This can be achieved through the creation of a drug, in which interferon gamma will be native, sterile, non-toxic, pyrogen-free and will contain a minimum amount of endotoxins.

At the end of the 20th century, studies of human immune interferon were carried out on very small amounts, which made it difficult to study the characteristics of the protein and use for therapeutic purposes. Interferon gamma was first obtained mainly by mitogenic induction of lymphocytes (RF patent No. 2092561). In 1981, a message appeared about a much more thorough, but still partial purification of the human immune interferon (Υ.Κ. Υίρ, K.N. Rapd, S. Itbap, app 1. Ubsek. RaSha1 rshtysabop apb sagasep / ayup ίί апitap datta ( 1 type) LjegGegop. Rgos Iab Asab δα υδΑ. 1981 MAGS; 78 (3): 1601-1605). This compound was obtained from cultures of lymphocytes stimulated by a combination of phytohemagglutin and phorbol and was purified by successive chromatographic separations. As a result of this procedure, a product with Mg 58 kDa was obtained.

Later, human immune interferon began to be obtained in very small quantities by translation of mRNA in oocytes, which acquired characteristics of the activity of human immune interferon.

Some time later, recombinant DNA technology appeared, in which a genetic construct was constructed, the basic elements of which are necessary for its replication and the possibility of its identification, in which the gene whose expression is necessary is cloned, due to the presence of regions specifically cleaved by restrictase enzymes in the genetic construct, cells are transformed with this construct recipient living organism. Recombinant DNA technology allowed to obtain the required protein in large quantities.

One of the first attempts to synthesize human gamma interferon (RF patent No. 2092561) was carried out as follows. 1) Mitogen stimulated production of human spleen cells or peripheral blood lymphocytes of the immune interferon. 2) A cell pellet was extracted from such a cell culture in the presence of a ribonuclease inhibitor to isolate all cytoplasmic RNA. 3) Fully messenger RNA (mRNA) in polyadenylated form was isolated on an oligo-AT column. This RNA was size fractionated using a sucrose density gradient and gel-electrophoresis in an acid-urea system. 4) RNA from 12δ to 18δ was converted into the corresponding single-stranded complementary DNA (cDNA), from which double-stranded DNA was obtained. After poly-bS crosslinking, it was included in a vector carrying one or more genetic markers. 5) The vectors thus obtained were used to transform bacterial cells. 6) The corresponding plasmid DNA was isolated from transforming clones and sequenced. 7) DNA with a certain sequence of bases was inserted into the corresponding expression vector with which the host cells were transformed.

When using column chromatography, a mRNA scatter (12δ-18δ) is obtained; therefore, it is more expedient to use a method that allows one to isolate a specific mRNA encoding interferon gamma. Moreover, in this case, two different vectors were used, which complicates the method and increases the time spent on obtaining the protein preparation. Obviously, such a method needs to be improved.

Moreover, in this case, the body for the expression of a gene encoding human interferon gamma was Bassyatotus ssgsuyas yeast. The expression of a foreign gene in yeast can be initiated by the 6'-side DNA sequence (promoter) from the yeast gene, located at the 5'-end of the non-yeast gene. In addition to the promoter, expression of the non-yeast gene in yeast requires a second yeast sequence located in the plasmid at the 3'-end of the non-yeast gene in order to ensure appropriate transcription termination and polyadenylation. This system must be simplified to save time and reagents.

In the methodology used by us, the above problems are resolved. One vector is used in the work, which ensures the synthesis of the necessary protein in E. soy cells, for which you do not need to modify the structure of the gene encoding interferon gamma. Antiviral activity was tested by a more sensitive and less laborious method.

There is evidence of another method for producing human gamma interferon (RF patent No. 2214832). 1. Synthesis of interferon gamma was induced in lymphocytes of human peripheral blood with staphylococcal enterotoxin B, after which the cells were washed with phosphate-buffered saline (PBS). 2. The mRNA was isolated using a sequentially lysis buffer containing sodium dodecyl sulfate and an RNase / protein splitter, 5 M # C1 and oligo (bT) cellulose, with which the mRNA was washed on a column. 3. Synthesis of cDNA was carried out with mRNA using M-MiLU reverse transcriptase, a set and protocol of Pallaaa Vulcis. 4. The required DNA fragment was ligated with plasmid RIS 18. 5. The E. coli cells of strain 1M 83 were transformed with a l-agar ligase mixture containing X-da1 and IPTG. 6. The required DNA fragment was ligated with a synthetic heteroduplex containing a ribosome binding site, and this fragment was inserted into the plasmid ptyu 315 (Shmelev V.A. et al., Molec. Genet. Microbe. Virusol. - 1995. - 1. - p. 9 -14), from which a fragment encoding thymosin was removed. 7. The obtained plasmid transformed E. coli cells of strain HB 2151 with plating on L-agar containing ampicillin. 8. Plasmid DNA was isolated. 9. Transformed with the obtained plasmid suitable strain E. soi (80 20050 or B 21) and cells of the transformed strain were cultured in normal medium. 10. Lysed cells and washed the aggregated protein. 11. Conducted chromatographic purification on a cation exchanger with simultaneous renaturation (column with cellulose KM-52). The recombinant human interferon gamma thus obtained consisted of 144 a.a., was devoid of the three Ν-terminal amino acids of the Suk-Tug-Suk mature protein, unlike the natural one, and had a different molecular weight (16.9 kDa) and p1 ( 10.36).

Using the technique described by us, one can obtain a native unprocessed recombinant human gamma interferon without methionine at the Ν-terminus.

The closest analogue of the invention is described in RF patent No. 2097428. This is a group of inventions: the recombinant plasmid rTTdKt2, which provides the synthesis of human gamma interferon in E. soi, a producer strain and a method for producing recombinant human gamma interferon. The plasmid rTTdKt2 was constructed on the basis of the plasmid pIS 19 containing the kanamycin resistance gene, the tryptophan promoter, followed by the human interferon gamma gene and transcriptional terminators ghpVTDg. This plasmid was introduced into the E. coli strain C600, which is a strain-producing agent of human interferon gamma. The method for producing interferon gamma used here includes culturing the cells of the producer strain in a medium with a low tryptophan content, lysing the cells, washing the aggregated protein, dissolving interferon gamma in 7 M urea with cetavlon, renaturation by ten-fold dilution with water, fractionating and precipitating interferon gamma sulfate ammonia and chromatography on an anion exchanger or optionally reverse phase high performance liquid chromatography.

The disadvantage of plasmid rTTdKt2 is that the expression of the human interferon gamma gene from the tryptophan promoter is triggered in tryptophan deficient media. In normal culture media, the activity of the tryptophan promoter is weakened (repressed). At the same time, the amount of tryptophan in the components of ordinary nutrient media (such as yeast extract, casein hydrolysates: tryptone, peptone) is not limited, therefore, the search or preparation of media with a lower content of tryptophan complicates the technology and increases the cost of the cultivation stage.

In addition, the use of recombinant human interferon gamma strain C600 E. as a producer strain does not fully meet the requirements for strain producers, in particular, on the productivity and stability of the synthesized recombinant proteins. A more productive E. coli strain, Bb 21, which has 1op and antTT mutations in protease genes, allows the production of non-proteinized recombinant proteins in large quantities.

The method for producing interferon gamma specified in RF patent No. 2097428 involves renaturation by tenfold dilution with water of interferon gamma dissolved in a 7 M urea solution, and then fractionation and precipitation of interferon gamma with ammonium sulfate. This is not technological, since operations with large volumes of solutions require a greater consumption of reagents (the same sulfate

- 2 023379 ammonium) and the use of large-scale equipment. Further, in the claims, chromatography on an anion exchanger is indicated, and interferon gamma has a positive charge at a pH of from 0 to 10 and cannot be adsorbed onto positively charged anion exchangers.

Regarding other stages of the work, namely, the process of isolating inclusion bodies and further protein extraction from them, other authors (US patent No. 4681930) proposed chemical lysis of the cells of the producer strain E. soi and simultaneous solubilization of interferon gamma from inclusion bodies with a solution of 7 M guanidine chloride ; remove insoluble components by centrifugation; Dilute the target fraction 10 times with pH 9.5 buffer and separate the resulting protein precipitate by centrifugation; fractionate the protein on silica gel (LiCe1-952 AC) and, finally, perform affinity chromatography on a sorbent with monoclonal antibodies.

The resulting product was in a solution of 20 mM sodium phosphate buffer containing 1 M NaCl and 1 M guanidine chloride (or 50% ethylene glycol), and was a protein with Mg 18 kDa homogeneous by electrophoresis. The yield of 25 g of cells was 8 mg (25-32% of the content in the cell extract). The biological activity of the drug in inhibiting the cytopathic effect was 10 ⁷ units / mg protein.

The result shows that the protein yield must be increased. Therefore, a modification of this method was proposed (patent No. 2132386). Cells were destroyed in the buffer solution, then a large mass of E. coli ballast cell proteins was separated by centrifugation of the cell extract from the insoluble target product located in the inclusion bodies, while zinc and copper salts were used to prevent proteolytic degradation of the target protein. The use for the solubilization of interferon gamma from bodies of inclusion of a urea denaturing agent less than guanidinochloride, a denaturing agent of the urea, provides more efficient protein renaturation (M. Ma18iata, Ό. Yoiaga, T. 8aka1 // Siet. Ryatt.Viy., 1992, ν. 40, No. 2, p. 550-552). The use of COOH-Biochrom K-1 sorbent for chromatographic purification significantly simplified and reduced the cost of the method compared to the use of an immune sorbent.

However, the method used in our experiments is even more advanced. Due to the fact that the cells were lysed not only in the buffer (different from the above), but also by ultrasound, they ensured more efficient destruction of the cells and the absence of the harmful effect of the substances that make up the buffer on the necessary protein. Moreover, in our work, inclusion bodies were washed with 0.2 M sodium deoxycholate 3 times, whereby lipopolysaccharides were removed, therefore, a pure pharmaceutical preparation was obtained, without any admixtures of bacterial endotoxins. To solubilize interferon gamma from inclusion bodies, we also used urea, but to obtain the purified protein, we used §-§periato8e, which is stable with a large spread of pH values (4-13) and greater binding ability.

The researchers proposed to modify the amino acid sequence of the interferon gamma protein, namely, to protect the C-terminal region of the molecule, since it is extremely saturated with sites for various proteases (RF patent No. 2105813). However, it is not known whether a positive effect will be observed in the patient if the molecule is long-lived. Moreover, if the drug is injected into the bloodstream, there is no need to protect interferon gamma from the effects of cellular proteases, since the molecule will not enter the cells, it will only interact with the corresponding receptor. Also, unlike the form of the drug for oral administration, it is not so important for the injectable form to be resistant to gastrointestinal proteases. In connection with the above reasons, our preparation does not contain a Ν-terminal methionine and any modifications at the C-terminus.

DETAILED DESCRIPTION OF THE INVENTION

A strain of Exxeysois B21 (PE3) -p1E-datta is proposed - a producer of human interferon gamma, collection number VKPM B-11012. The strain is characterized by antibiotic resistance - ampicillin.

The obtained highly purified preparation of native human gamma interferon has a high specific biological activity (2,000,000 units per 50 μg) and stability. According to polyacrylamide gel electrophoresis with the addition of sodium dodecyl sulfate, at least 95% of the main substance of the drug is in the form of a monomer, and in the dimeric form - not more than 5%. The resulting preparation only reacts with antibodies to interferon gamma (Rerto1esy company, catalog number 500-M90), is stable in the pH range from 4.5 to 10. Specific antiviral activity is at least 2-10 ⁶ IU / mg.

A concentrated solution of interferon gamma is diluted to a therapeutic concentration (10 to 100 μg / ml) with an aqueous buffer solution containing stabilizing additives. The liquid semi-finished product is lyophilized and stored at a temperature not exceeding + 10 ° C for 1.5 years.

The resulting preparation is non-toxic, pyrogen-free when tested in animals, has controlled antiviral activity when tested in a culture of human lung carcinoma cells (A549) infected with EMC virus (from 50,000 IU / ml solution to 1 million IU / ml solution), does not cause adverse reactions in test animals; the drug quickly, within a few seconds, dissolves in water and physiological saline for injection, the solution remains transparent at room temperature for at least 48 hours

This drug was obtained through a series of experiments.

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Example 1. Obtaining a gene encoding interferon gamma human (cDNA).

1.1. Monocyte cells of human peripheral blood (MCPC) were obtained by centrifugation of heparinized blood in a density gradient of HCO11-Rac | Is (Wowit, 1964): resuspended cells (10 ml) were carefully layered on 4 ml of HCOI-Rasck solution (Pbtasha Vulcis, Sweden) and centrifuged at room temperature for 30 min at 400 d on a benchtop centrifuge. The cell ring (monocytes and lymphocytes) formed after centrifugation was carefully taken with a pipette and resuspended in a 20-fold volume of PB§. MCPC was purified three times by centrifugation in a solution of PB§ at room temperature for 10 min at 400 d in a benchtop centrifuge. After the third wash, the cells were resuspended in 1 ml of PB§. Lymphocytes were cultured in PPM1 1640 medium containing 10% PB§.

1.2. The synthesis of mRNA encoding human interferon gamma in lymphocytes was stimulated with a solution of PMA (pryuglobatum aceridae, §1 §ta, USA), and allostimulation of homologous PBMCs obtained from different individuals was used. For this, 10 ⁷ MCPCs obtained from 3 independent individuals were mixed in one falcon, washed 1 time with PB§ and resuspended in 1 ml of PPM1 1640 medium containing 1% PBB, PMA was added to a concentration of 25 ng / ml (stock solution 1 mg / ml in ΌΜδΘ) and incubated for 6-8 hours in a CO ₂ incubator at + 37 ° C. After incubation, the cells were collected by centrifugation, washed in PB8 and used to isolate RNA.

1.3. RNA was isolated using a method based on the use of guanidine isothiocyanate, followed by purification by ultracentrifugation in a density gradient of cesium chloride.

For this, stimulated PBMCs were resuspended in 300 μl of physiological saline (NaCl), 1 ml of a solution containing 5.5 M guanidine isothiocyanate, 50 mM sodium acetate, 50 mM EDTA, 0.5% sarcosyl and 7 μl β-mercaptoethanol was added to them. The mixture was stirred and 1 ml of a solution containing 5.7 M cesium chloride, 25 mM sodium acetate, 50 mM EDTA was added. The resulting suspension was layered in 1.5 ml of a solution containing 5.7 M cesium chloride, 25 mM sodium acetate, 50 mM EDTA, placed in a centrifuge tube of the δν65 rotor (Westap, USA). Centrifugation was carried out at 100000 d for 18 hours at + 20 ° C. After centrifugation, the supernatant was drained, and the precipitate containing RNA encoding interferon-gamma was washed 3 times with 70% ethanol and dried overnight at -20 ° C. RNA was stored dry or under 70% ethanol at -70 ° C.

1.4. To obtain the cDNA sequence encoding interferon gamma, an RT-PCR reaction was performed. RNA derived from 10 ⁷ -10 ⁸ stimulated PBMC cells was dissolved in 20 μl of water. 5 μl of total RNA and 0.5 μg oido (bT) of ₁₈ primers were used for the reverse transcription (RT) reaction. The reaction was carried out in accordance with the instructions for the collection of ReuPAL Ρίτκΐ δίππιά SOYA 8uye518 Κίΐ (Reptep! Ak, Lithuania). The first (+) DNA strand resulting from RT was used as a template in the polymerase chain reaction (PCR) to amplify a gene encoding interferon gamma. For this, 5 μl of RT-PCR and 0.2 pM of each primer were taken (ΙΡΝ-γ-cr: 5'-STSOSTAOSSASSASSO OSSTTOASSTTTOST-3 'and ΙΡΝ-γ-5'-OTAAOSTATSATTSSTSTASTSTSTAAAST-3'). The reaction was carried out under standard conditions for the Tatz polymer (Retteyak, Lithuania) according to the program: 95 ° С - 10 s, 58 ° С - 10 s, 72 ° С - 40 s for 35 cycles, 72 ° С - 2 min for completion end sequences.

The result of PCR was analyzed by electrophoresis on a 1% agarose gel.

Example 2. The creation of plasmid DNA (ρΙΕ-Datta) containing a gene encoding human interferon gamma.

2.1. According to the method described in example 1, a gene was synthesized encoding human interferon gamma: received human PBMC (monocytes and lymphocytes); lymphocytes were cultured in PPM1 1640 medium containing 10% PBδ; stimulated the synthesis of mRNA encoding human interferon gamma by lymphocytes; isolated this mRNA; synthesized cDNA (necessary gene) on the mRNA matrix.

2.2. The cDNA of the gene encoding human interferon gamma was cloned in the plasmid vector pOetTeac (Rgoted, USA) using the reagents of this kit.

POetTEaku vector contains the following components: promoter for T7 phage polymerase, which provides control of gene expression level, 1ac-operator to provide controlled expression of the gene encoding the desired protein, binding site to the ribosomal complex (ΚΡδ), start codon for translation of the cloned fragment, DNA fragment encoding polyhistidine, which is in the translation frame, which ensures the expression of the required nucleotide sequence in the form of a protein fused with polyhistidine, for the convenience of its further characterization. sources using chromatography, the nucleotide sequence encoding the Y5 epitope in the linker, which provides the synthesis of the Y5 epitope crosslinked with the desired protein; monoclonal antibodies to this epitope make it possible to analyze the expression of any inserted gene using immunoblotting, a site for the TEU protease, which allows to obtain a protein in the form of a separate amino acid sequence, without polyhistidine and Y5 epitope, a fragment of mountains that provides the optimal copy number of the plasmid, pBK322ot1 to initiate replication, 1ac1 is a lactose repressor to ensure the operation of the 1ac operon, an ampicillin resistance site for the selection of clones containing this vector.

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The result of PCR was precipitated with ethanol in the presence of 2 M ammonium acetate, centrifuged at 10000 d for 5 min, the precipitate was washed with 70% ethanol, dried and dissolved in 10 μl of water.

For the ligation reaction, 3 μl of the cDNA solution, 1 μl of the solution of the prepared vector rCetTeacu, 5 μl of x2 ligation buffer and 1 μl of T4 ligase were taken. The reaction was carried out at + 20 ° C for 2 hours

After this, the mixture was heated at + 95 ° C for 10 min and purified from salts by dialysis on nitrocellulose filters with a pore diameter of 0.025 μm (MNNroge, USA). Dialysis was performed against a solution containing 0.5 mM EDTA in 10% glycerol for 10 minutes.

Example 3. The creation of a strain of E. coN for amplification of plasmid DNA containing a gene encoding human interferon gamma.

3.1. By the method described in example 2, a gene was synthesized that encodes human interferon gamma: received human PBMC (monocytes and lymphocytes); lymphocytes were cultured in PPM1 1640 medium containing 10% PB8; stimulated the synthesis of mRNA encoding human interferon gamma by lymphocytes; isolated this mRNA; synthesized cDNA (the necessary gene) on the mRNA matrix and obtained plasmid DNA of the rGE-datta: the synthesized gene (cDNA on the mRNA matrix) was ligated into the rCetTeac vector.

3.2. Transformed the cells of E.eo strain ΌΗ10Β / Κ. (E ^- tagA, D (TGG-Y8YRM§-tsgVS), f80N / ac2DM 15, D1acX74, Neo-HesA1, epNA1, agaE139, D (aha, 1ey) 769, §a1I, da1KX ^- , hrc, irc) plasmid DNA rGE-Datta by electroporation using a generator of high-voltage pulses GVI-1 (St. Petersburg State Technical University, St. Petersburg). For this, 1 μl of the dialyzed ligase mixture was added to 12 μl of competent cells, placed between the electrodes of the paired cell and treated with a current pulse of 1 kV per 1 mm and duration of 4 ms.

3.3. After transformation, the cells were placed in 1 ml of 8OC medium (2% bacto-tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl ₂ , 10 mM MgSO ₄ , 20 mM glucose) and incubated for 40 min at + 37 ° C.

3.4. E.coH cells were screened for plasmids in a selective medium containing LBar, 100 μg / ml ampicillin, 5 mM 1RTS, 20 μg / ml X-da1. 10 colonies of E. col, developing a white color on the chromogenic substrate X-da1, were checked for the presence of a gene encoding interferon gamma by PCR. The reaction conditions were the same as in the RT-PCR reaction (see example 1).

3.5. Screening analysis was performed by electrophoresis on a 1% agarose gel. Colonies of E. coN cells containing plasmids with the insert of a gene encoding interferon gamma were used as a night seed for the subsequent isolation of plasmid DNA.

3.6. Isolation of plasmid DNA was carried out using the set of Нνί / agN Mnerger ΌΝΑ Rshtisayoi §u81et (Rgoteda, USA). Purified recombinant plasmid DNA (p1E-Datta) was checked by restriction analysis and sequencing.

Example 4. The creation of a strain of E. coN - producer of interferon gamma human.

4.1. By the method described in example 3, a gene was synthesized that encodes human interferon gamma: received human PBMC (monocytes and lymphocytes); lymphocytes were cultured in PPM1 1640 medium containing 10% PB8; stimulated the synthesis of mRNA encoding human interferon gamma by lymphocytes; isolated this mRNA; synthesized cDNA (the necessary gene) on the mRNA matrix, obtained plasmid DNA of the rGE-dataset: the synthesized gene (cDNA on the mRNA matrix) was ligated into the rCetTeac vector and received a large amount of plasmid DNA of rGE-datta, which could now be used to synthesize the human gamma interferon protein : transformed E. col cells of the HH10B / P strain of plasmid DNA p1E-datt; incubated in 8 ° C-medium, E.coN cells were screened for plasmids in a selective medium; plasmid DNA was isolated.

4.2. Prepared E. col cells of strain B21 (HE3) with the genotype P-otrT Y8YGB (hB-tB-) §-1 Hst gpe131 (SEE3) as follows. Cells were incubated at + 37 ° C overnight in 5 ml of L-broth containing 1% tryptone, 1% yeast extract and 1% sodium chloride. The culture was diluted with fresh L-broth 50-100 times and grown on a shaker at + 37 ° C to an optical density of 0.2-0.3 at a wavelength of 590 nm. Upon reaching an optical density of more than 0.3, the culture was bred with fresh L-broth to an optical density of 0.1 and grown for 30 minutes. Transferred 100 ml of culture to a sterile centrifuge tube and pelleted cells at + 4 ° C for 5000 d for 10 min. The supernatant was drained, the cells were resuspended in 50 ml of 0.1 M CaCl ₂ cooled on ice. The cells were incubated for 20 min on ice. Cells were pelleted for 10 min at 5000 rpm, + 4 ° C. The supernatant was drained, the cells were resuspended in 3 ml of 0.1 M CaC12, cooled on ice.

4.3. For the expression of the gene encoding human interferon gamma, E. coN cells of strain B21 [HE3] were transformed, having 1o- and srT mutations by protease genes and allowing to obtain non-proteolyzed recombinant proteins in large quantities created by the plasmid by electroporation.

3 ml of competent cells were transferred to a cold tube and 5-10 μg of p1E-datt plasmid DNA was added. Incubated on ice for 1 h. Transferred the tube to + 42 ° C and kept for 5 minutes. The cells were transferred to a flask with 100 ml of L-broth heated to + 37 ° C without antibiotic and incubated at + 37 ° C in

- 5 023379 for 1-1.5 hours. Then the cells were used directly for inoculation into the fermenter.

The obtained plasmid pGE-dash after transformation into competent cells of the strain E. coB B21 (HE3) provides a high level of biosynthesis of recombinant human gamma interferon determined by electrophoresis in 15% SDS page-SDS.

Example 5. Obtaining human gamma interferon in E. soy cells by inducing protein synthesis with 0.2% lactose according to the Stuyder method.

5.1. By the method described in example 4, a gene was synthesized that encodes human interferon gamma: received human PBMC (monocytes and lymphocytes); lymphocytes were cultured in medium ΚΡΜΙ 1640 containing 10% PB8; stimulated the synthesis of mRNA encoding human interferon gamma by lymphocytes; isolated this mRNA; synthesized cDNA (the necessary gene) on the mRNA matrix, obtained plasmid DNA of the rGE-dash: the synthesized gene (cDNA on the mRNA matrix) was ligated into the pOeshTEak vector and received a large amount of plasmid DNA of the rGE-dash, which could now be used to synthesize human gamma interferon protein : E.soy cells of the strain ΌΗ10Β / Κ of the plasmid DNA ρΙΕ-datt were transformed; incubated in 8 ° C-medium, E. soy cells were screened for plasmids in a selective medium; plasmid DNA was isolated, E. coli strain was obtained - producer of human gamma interferon: E. coli cells of strain B21 were prepared for transformation of plasmid DNA; E. coli cells of strain B21 [HE3] were transformed with the created plasmid pGE; transferred cells to L-broth with a solution temperature of + 37 ° C without antibiotic; incubated at + 37 ° C for 1-1.5 hours; seeded transformed cells into a fermenter.

5.2. For auto-induction of expression according to the Stuyder method (81ib1et, 2005), ΡΥΡ5052 medium was used, consisting of 1% peptone (Syso. USA), 0.5% yeast extract (O & Co, USA), 50 mM Na ₂ NRA ₄ , 50 mM K ₂ NRA ₄ , 25 mM (OH ₄ ) ₂ §O ₄ , 2 mM Md§O ₄ , 0.5% glycerol, 0.05% glucose and 0.2% lactose.

On Wednesday ΡΥΡ-5052 containing ampicillin at a concentration of 100 μg / ml, a single colony of the producer strain was inoculated. After it was fermented at + 37 ° C in a thermostatically controlled rotary type shaker at 250 rpm for 20 hours until there was no significant change in optical density at a wavelength of 600 nm for 1 hour, an aliquot of cells was then selected for analysis by electrophoresis, and the remaining biomass was precipitated by centrifugation at 9000d and left to be stored at -70 ° C for subsequent protein isolation.

Example 6. Obtaining a pure pharmaceutical preparation of a native recombinant human gamma interferon without bacterial endotoxin impurities.

6.1. By the method described in example 5, a gene was synthesized that encodes human interferon gamma: received human PBMC (monocytes and lymphocytes); Lymphocytes were cultured in medium ΚΡΜΙ 1640 containing 10% LB8; stimulated the synthesis of mRNA encoding human interferon-gamma by lymphocytes; isolated this mRNA; synthesized cDNA (the necessary gene) on the mRNA matrix, obtained plasmid DNA of the rGE-probe: the synthesized gene (cDNA on the mRNA matrix) was ligated into the pOeshTEak vector and received a large amount of plasmid DNA of p1E-datta, which could now be used to synthesize the human gamma interferon protein : E. coli cells of the OH10B / P strain of plasmid DNA p1E-datt were transformed; incubated in 8 ° C-medium, E. soy cells were screened for plasmids in a selective medium; plasmid DNA was isolated, E. coli strain was obtained - producer of human gamma interferon: E. coli cells of strain B21 (HE3) were prepared for transformation of plasmid DNA; transformed E. coli cells of strain B21 [HE3] with the created plasmid p1E-datta; transferred cells to L-broth with a solution temperature of + 37 ° C without antibiotic; incubated at + 37 ° C for 1-1.5 hours; transformed cells were seeded into a fermenter, protein was synthesized: expression of a gene encoding interferon gamma, 0.2% lactose was induced by the Stuyder method.

6.2. The interferon gamma protein was isolated as a part of inclusion bodies from E. soy cells by cell lysis. Cells were resuspended in lysis buffer containing 20 mM Tris-HCl pH 7.5, 5 mM EDTA and 1 mM phenoxymethylsulfonyl fluoride based on 1 g of cells 5-7 ml of buffer. The cell suspension was sonicated 7 times for 30 s with an interval of 30 s (ultrasound frequency is 22 kHz). The lysate was centrifuged for 10 min at + 4 ° C, 5000 d. The supernatant was drained, a solution of 1 M urea was added to the precipitate at the rate of 10 ml per 1 g of cells, and intensively mixed. Repeated centrifugation. The supernatant was drained, the precipitate was resuspended in a solution of 2 M urea of the same volume. Repeated centrifugation. The supernatant was drained.

6.3. The inclusion bodies were washed 3 times with 0.2 M sodium deoxycholate 3 times, whereby the lipopolysaccharides were removed, therefore subsequently (see Example 7, paragraphs 7.2-7.6) a pure pharmaceutical preparation was obtained, without the admixtures of bacterial endotoxins.

7. Obtaining purified unprocessed interferon gamma human without methionine at the Ν-end.

7.1. By the method described in example 6, a gene was synthesized that encodes human interferon gamma: received human PBMC (monocytes and lymphocytes); lymphocytes were cultured in medium ΚΡΜΙ 1640 containing 10% PB8; stimulated the synthesis of mRNA encoding human interferon gamma by lymphocytes; isolated this mRNA; synthesized cDNA (the necessary gene) on the mRNA matrix, obtained plasmid DNA of the rGE-dash: the synthesized gene (cDNA on the mRNA matrix) was ligated into the rCetTeac vector and received a large amount of plasmid DNA of ρΙΕ-dash, which could now be used for synthesis human gamma interferon protein: E.soy cells of the ΌΗ10ΌΗ / Κ strain of the ρΙΕ-datt plasmid DNA were transformed; incubated in 8 ° C-medium, E. soy cells were screened for plasmids in a selective medium; plasmid DNA was isolated, E. coli strain was obtained - producer of human gamma interferon: E. coli cells of strain B21 (PE3) were prepared for transformation of plasmid DNA; transformed E. coli cells of strain B21 [OE3] with the created plasmid pGE-datta; transferred cells to L-broth with a solution temperature of + 37 ° C without antibiotic; incubated at + 37 ° C for 1-1.5 hours; seeded transformed cells into a fermenter, synthesized a protein: induced expression of a gene encoding interferon gamma, 0.2% lactose according to the Stuyder method, isolated inclusion bodies from E. soy cells: cells were lysed, lipopolysaccharides were removed: inclusion bodies were washed with 0.2 M sodium deoxycholate 3 times, by which resulted in a very pure pharmaceutical preparation, without the admixtures of bacterial endotoxins.

7.2. The precipitate was dissolved in a buffer containing 0.1 M ammonium acetate, pH 7.5 and 8 M urea, of the same volume. Centrifuged. The supernatant was a solution of interferon gamma of about 60-70% electrophoretic purity.

7.3. After containing in an 8 M urea solution, the protein was treated with a TEU protease to cleave the Ν-terminal methionine, a site for a TEU protease and a polyhistidine sequence and passed through a 10 kDa cut-off pore membrane to separate these amino acid sequences from the gamma interferon protein.

7.4. Spent refolding interferon gamma human protein. We placed interferon-gamma without the кон-terminal methionine in the buffer for refolding (0.1 M Tg pH 8.0, 0.2 mM EDTA) with 0.5 M L-Arginine. Dissolved inclusion bodies aggregated depending on the amount of arginine. Aggregation was observed at a wavelength of 500 nm. The buffer was stirred at 10 ° C, added three times in 10 ml ΙΒ with an interval of 2 hours. The solution was incubated for 36-48 hours, without stirring, at 10 ° C. At higher temperatures, aggregation prevailed.

7.5. The renatured protein was desalted with a solution of 20 mM Tp5-HC1 pH 8.0 containing 100 mM urea. Dialysis was carried out at 10 ° C until a conductivity of 3-3.7 μS was achieved. The solution was centrifuged at 10,000 gt for 10 min, and the supernatant was collected.

7.6. Chromatographic purification of the obtained solution of interferon gamma human without Ν-terminal methionine was carried out. About 1200 ml of desalted supernatant was placed on a 20 ml 8 Zeryagoka column, equilibrated with 20 mM Tn5-HC1 pH 8.0. The column was washed with 200 ml of 50 mM TgF pH 8.0, the protein was eluted with 160 ml of a linear gradient of 50 mM TgF pH 8.0 1 M No. C1. The fraction of human gamma-purified interferon 8 at Zeriagok without the кон-terminal methionine was placed on an 8-100 column (2.5x80 cm), previously equilibrated with phosphate buffer. Fractions were analyzed using 8E8-PACE. The column was calibrated using 10 mg bovine serum albumin, ovalbumin and a soybean trypsin inhibitor.

Claims (2)

CLAIM 1. The strain Exceis soy B21 (PE3) -p1E-Datta is a producer of human gamma interferon, VKPM B-11012. 2. A method of obtaining a preparation of recombinant human gamma interferon, which consists in cultivating a recombinant strain E. soi, a producer of interferon gamma in a nutrient medium, isolating the aggregated protein, followed by its renaturation and purification from impurities, characterized in that as a recombinant strain use the E. coli strain according to claim 1, after cultivation, the expression of a gene encoding human gamma-interferon is induced, the aggregated protein is isolated as a result lysis of bacterial cells with sequential use of a buffer of 20 mM Tris-HC1 pH 7.5, 5 mM EDTA and 1 mM phenoxymethylsulfonyl fluoride and ultrasound, washing the inclusion bodies three times with 0.2 M sodium deoxycholate, followed by treatment with TEU protease and passing through a membrane filter, and renaturation is carried out using 8 M urea solution in a buffer containing 0.1 M TGY pH 8.0, 0.2 mM EDTA, 0.5 M L-arginine, after which the renatured protein is desalted with a solution of 20 mM TP5-HC1 pH 8.0 containing 100 mM urea, purification of the obtained solution The protein solution is carried out on an 8-8 column 5 and 8-100 column (2.5x80 cm) using appropriate buffers; a highly purified solution of the obtained unprocessed non-methionine native recombinant human gamma is mixed with a physiologically acceptable carrier.